技术领域Technical Field
本申请涉及生物技术领域，具体涉及一种用于酶促DNA合成的固相载体及其制备方法、用途。The present application relates to the field of biotechnology, and in particular to a solid phase carrier for enzymatic DNA synthesis and a preparation method and use thereof.
背景技术Background Art
酶促DNA合成是一种基于末端脱氧核苷酸转移酶的新一代DNA合成技术。相对于传统的化学DNA合成方法，酶促DNA合成更加绿色环保，且具有更长的合成长度潜力。类似于传统的化学DNA合成技术，酶促DNA合成过程需要经过冲洗掉上一步的反应溶液，保证接下来的反应进行。因此需要固相载体表面对引发链进行偶联，在引发链的存在条件下，加入封闭3’-OH端的碱基单体，基于末端脱氧核苷酸转移酶的作用，实现单碱基的偶联后终止，再通过亚硝酸根的脱保护作用，继续延伸下一个碱基，从而自由的碱基延伸组合，最终完成的DNA合成。为了保证末端脱氧核苷酸转移酶在固相载体表面的工作，提高偶联效率，固相载体需要较低的反应位阻，更强的舒展能力。但现有技术中的固相载体的反应位阻较高，舒展能力也较差。Terminal deoxynucleotidyl transferase A new generation of DNA synthesis technology. Compared with traditional chemical DNA synthesis methods, enzymatic DNA synthesis is more environmentally friendly and has the potential for longer synthesis length. Similar to traditional chemical DNA synthesis technology, the enzymatic DNA synthesis process needs to wash away the reaction solution of the previous step to ensure the next reaction. Therefore, it is necessary to couple the initiator chain on the surface of the solid phase carrier. Under the condition of the presence of the initiator chain, a base monomer with a closed 3'-OH end is added. Based on the action of the terminal deoxynucleotidyl transferase, the coupling of a single base is terminated, and then the next base is extended through the deprotection of nitrite, thereby freely extending the base combination and finally completing the DNA synthesis. In order to ensure the work of the terminal deoxynucleotidyl transferase on the surface of the solid phase carrier and improve the coupling efficiency, the solid phase carrier needs lower reaction steric hindrance and stronger stretching ability. However, the reaction steric hindrance of the solid phase carrier in the prior art is high and the stretching ability is also poor.
因此，目前用于酶促DNA合成的固相载体仍有待改进。Therefore, the solid phase supports currently used for enzymatic DNA synthesis still need to be improved.
发明内容Summary of the invention
针对现有技术存在的不足，本发明目的是提供一种用于酶促DNA合成的固相载体及其制备方法、用途，以解决上述背景技术中提出的问题。In view of the deficiencies in the prior art, the present invention aims to provide a solid phase carrier for enzymatic DNA synthesis and a preparation method and use thereof, so as to solve the problems raised in the above background technology.
在本发明的一个方面，本发明提供了一种用于酶促DNA合成的固相载体，所述固相载体包括水凝胶骨架材料、功能性材料、交联剂、添加剂以及引发链；所述水凝胶骨架材料包括丙烯酸酯类高分子材料；所述添加剂包括增强剂、增塑剂以及光引发剂；其中所述水凝胶骨架材料占各组分总质量的10%-35%。In one aspect of the present invention, the present invention provides a solid phase carrier for enzymatic DNA synthesis, wherein the solid phase carrier comprises a hydrogel skeleton material, a functional material, a cross-linking agent, an additive and an initiator chain; the hydrogel skeleton material comprises an acrylate polymer material; the additive comprises a reinforcing agent, a plasticizer and a photoinitiator; wherein the hydrogel skeleton material accounts for 10%-35% of the total mass of each component.
进一步地，所述水凝胶骨架材料经过活化处理并偶联所述引发链。Furthermore, the hydrogel skeleton material is activated and coupled to the initiator chain.
进一步地，所述水凝胶骨架材料包括丙烯酸甲酯、丙烯酸乙酯、丙烯酸正丙酯、丙烯酸正丁酯、甲基丙烯酸羟乙酯、聚乙二醇二丙烯酸酯、甲基丙烯酸羟丙酯、甲基丙烯酸羟丁酯中的至少之一。Furthermore, the hydrogel skeleton material includes at least one of methyl acrylate, ethyl acrylate, n-propyl acrylate, n-butyl acrylate, hydroxyethyl methacrylate, polyethylene glycol diacrylate, hydroxypropyl methacrylate, and hydroxybutyl methacrylate.
进一步地，所述功能性材料包括含有环氧基、乙烯基、伯胺、巯基、羰基、羟基、羧基中的至少之一的化合物。Furthermore, the functional material includes a compound containing at least one of an epoxy group, a vinyl group, a primary amine, a thiol group, a carbonyl group, a hydroxyl group, and a carboxyl group.
更进一步地，所述功能性材料包括甲基丙烯酸缩水甘油酯。Furthermore, the functional material includes glycidyl methacrylate.
进一步地，所述交联剂包括乙二醇二丙烯酸酯。Furthermore, the cross-linking agent includes ethylene glycol diacrylate.
进一步地，所述增强剂包括N-乙烯基吡咯烷酮。Furthermore, the enhancer includes N-vinyl pyrrolidone.
进一步地，所述增塑剂包括乙二醇。Furthermore, the plasticizer includes ethylene glycol.
进一步地，所述光引发剂包括2-羟基-4’-(2-羟乙氧基)-2-甲基苯丙酮以及2-羟基-2-甲基苯丙酮中的至少之一。Furthermore, the photoinitiator includes at least one of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone and 2-hydroxy-2-methylpropiophenone.
进一步地，所述水凝胶骨架材料是经过表面改性后进行所述活化处理的，所述表面改性包括在所述水凝胶骨架材料中引入氨基、羧基、环氧基、巯基以及乙烯基基团中的至少之一。Furthermore, the hydrogel skeleton material is subjected to the activation treatment after surface modification, and the surface modification comprises introducing at least one of an amino group, a carboxyl group, an epoxy group, a thiol group and a vinyl group into the hydrogel skeleton material.
进一步地，所述固相载体进一步包括溶剂。Furthermore, the solid phase carrier further comprises a solvent.
更进一步地，所述溶剂包括水。Furthermore, the solvent includes water.
进一步地，形成所述固相载体的原料中，所述水凝胶骨架材料、所述功能性材料、所述交联剂、所述添加剂与所述溶剂的体积比为（3-10）：（2-5）：（0.1-1）：（5-10）：（4-8）。Furthermore, in the raw materials forming the solid phase carrier, the volume ratio of the hydrogel skeleton material, the functional material, the cross-linking agent, the additive and the solvent is (3-10): (2-5): (0.1-1): (5-10): (4-8).
进一步地，形成所述固相载体的原料中，所述添加剂中所述增强剂、所述增塑剂与所述光引发剂的体积比为（1-5）：（3-6）：（0.1-1）。Furthermore, in the raw material for forming the solid phase carrier, the volume ratio of the reinforcing agent, the plasticizer and the photoinitiator in the additive is (1-5): (3-6): (0.1-1).
在本发明的另一方面，本发明提供了一种制备所述固相载体的方法，所述方法包括以下步骤：将所述水凝胶骨架材料、所述功能性材料、所述交联剂、所述添加剂与所述溶剂混合后倒入模具中，在光照条件下照射得到水凝胶骨架；对所述水凝胶骨架进行引发链偶联得到所述固相载体；其中，所述光照条件包括UV照射；光照时间包括8-12分钟。In another aspect of the present invention, the present invention provides a method for preparing the solid phase carrier, which comprises the following steps: mixing the hydrogel skeleton material, the functional material, the cross-linking agent, the additive and the solvent, pouring the mixture into a mold, and irradiating the mixture under light conditions to obtain a hydrogel skeleton; initiating chain coupling of the hydrogel skeleton to obtain the solid phase carrier; wherein the light conditions include UV irradiation; and the light irradiation time includes 8-12 minutes.
进一步地，进行所述引发链偶联之前，对所述水凝胶骨架表面进行改性处理得到改性水凝胶，所述改性处理包括用亲水改性化合物对所述水凝胶骨架表面进行一次或多次处理。Furthermore, before the initiator chain coupling is performed, the surface of the hydrogel skeleton is modified to obtain a modified hydrogel, and the modification comprises treating the surface of the hydrogel skeleton with a hydrophilic modification compound once or multiple times.
更进一步地，所述亲水改性化合物包括支化聚乙烯亚胺、聚谷氨酸以及聚乙二醇衍生物的至少之一。Furthermore, the hydrophilic modification compound includes at least one of branched polyethyleneimine, polyglutamic acid and polyethylene glycol derivatives.
进一步地，本方法进一步包括对所述改性水凝胶进行活化处理后进行所述引发链偶联，所述活化处理包括用活化化合物处理所述改性水凝胶；所述活化化合物包括碳二亚胺类羧基活化剂和NHS活化脂中间物；其中，所述碳二亚胺类羧基活化剂包括1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐和二环己基碳二亚胺中的至少之一；所述NHS活化脂中间物包括N-羟基硫代琥珀酰亚胺钠盐和N-羟基琥珀酰亚胺中的至少之一。Furthermore, the method further comprises performing an activation treatment on the modified hydrogel before performing the initiator chain coupling, wherein the activation treatment comprises treating the modified hydrogel with an activating compound. ; The activating compound includes a carbodiimide carboxyl activator and an NHS-activated lipid intermediate; wherein the carbodiimide carboxyl activator includes at least one of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and dicyclohexylcarbodiimide; and the NHS-activated lipid intermediate includes at least one of N-hydroxysulfosuccinimide sodium salt and N-hydroxysuccinimide.
进一步地，所述引发链偶联包括：将经过所述活化处理的改性水凝胶浸入链霉亲和素偶联溶液中过夜，而后再浸入引发链偶联溶液中，得到所述固相载体。Furthermore, the initiator chain coupling comprises: immersing the modified hydrogel after the activation treatment in a streptavidin coupling solution overnight, and then immersing it in an initiator chain coupling solution. , to obtain the solid phase carrier.
进一步地，所述链霉亲和素偶联溶液中包括链霉亲和素。Furthermore, the streptavidin coupling solution includes Streptavidin.
进一步地，所述引发链偶联溶液包括引发链。Furthermore, the initiator chain coupling solution comprises Initiate chain.
更进一步地，所述引发链包括5’端生物素标记的DNA序列。Furthermore, the priming strand includes a DNA sequence labeled with biotin at the 5' end.
在本发明的又一方面，本发明提供了一种利用所述固相载体合成DNA的方法，所述方法包括以下步骤：裁剪所述固相载体并利用其进行DNA合成反应和脱保护反应，不断重复进行所述合成反应与所述脱保护反应循环，合成目标序列；对经所述合成反应与所述脱保护反应循环后的固相载体进行解离反应；以所述解离反应得到的解离产物为模板进行PCR扩增；对所述PCR扩增得到的产物进行琼脂糖凝胶电泳，回收目标电泳条带并对其进行测序分析。In another aspect of the present invention, the present invention provides a method for synthesizing DNA using the solid phase carrier, the method comprising the following steps: cutting the solid phase carrier and using it to perform DNA synthesis reaction and deprotection reaction, continuously repeating the synthesis reaction and the deprotection reaction cycle to synthesize the target sequence; performing a dissociation reaction on the solid phase carrier after the synthesis reaction and the deprotection reaction cycle; performing PCR amplification using the dissociation product obtained by the dissociation reaction as a template; performing agarose gel electrophoresis on the product obtained by the PCR amplification, recovering the target electrophoresis band and performing sequencing analysis on it.
进一步地，所述合成反应包括：将裁剪的所述固相载体浸入到DNA酶促合成反应液中，在30℃-45℃的温度条件下反应；其中，所述DNA酶促合成反应液包括末端脱氧核苷酸转移酶、末端保护碱基单体、氯化钴、PBS和水。Furthermore, the synthesis reaction comprises: immersing the cut solid phase carrier into a DNA enzymatic synthesis reaction solution, reacting at a temperature of 30°C-45°C ; wherein the DNA enzymatic synthesis reaction solution includes terminal deoxynucleotidyl transferase, terminal protection base monomer, cobalt chloride, PBS and water.
进一步地，所述脱保护反应包括：取出经所述合成反应的所述固相载体，冲洗后浸入到脱保护反应液中，后取出所述固相载体并进行冲洗；而后重复进行所述合成反应与所述脱保护反应；其中，所述脱保护反应液包括亚硝酸钠和冰乙酸。Furthermore, the deprotection reaction comprises: taking out the solid phase carrier after the synthesis reaction, rinsing it and immersing it in a deprotection reaction solution, Then the solid phase carrier is taken out and rinsed; and then the synthesis reaction and the deprotection reaction are repeated; wherein the deprotection reaction solution includes sodium nitrite and glacial acetic acid.
进一步地，在进行所述解离反应之前，进一步包括：将经所述合成反应与脱保护反应循环后的固相载体浸入到PolyA合成反应液中，在30℃-45℃的温度条件下反应令引发链的3’端合成PolyA片段；其中，所述PolyA合成反应液包括末端脱氧核苷酸转移酶、无保护的dATP单体、缓冲液和水。Furthermore, before the dissociation reaction is performed, the method further comprises: immersing the solid phase carrier after the synthesis reaction and the deprotection reaction cycle into the PolyA synthesis reaction solution, and reacting at a temperature of 30°C to 45°C. The 3' end of the initiator chain is made to synthesize a PolyA fragment; wherein the PolyA synthesis reaction solution comprises terminal deoxynucleotidyl transferase, unprotected dATP monomer, buffer and water.
进一步地，所述解离反应包括：将所述合成反应与所述脱保护反应循环后的所述固相载体浸入到纯水中，在80℃-100℃的温度条件下反应得到解离产物。Furthermore, the dissociation reaction comprises: immersing the solid phase carrier after the synthesis reaction and the deprotection reaction cycle in pure water, reacting at a temperature of 80°C-100°C The dissociation product is obtained.
本发明至少具有以下有益效果的至少之一：The present invention has at least one of the following beneficial effects:
1、本发明的固相载体，采用水凝胶作为骨架材料，溶胀而不溶解，具有更强的舒展能力；1. The solid phase carrier of the present invention uses hydrogel as the skeleton material, which swells but does not dissolve and has stronger stretching ability;
2、本发明的固相载体，采用水凝胶作为骨架材料，在其表面接枝功能性基团，可用于酶促DNA合成，有效降低了酶促反应过程的空间位阻，合成效率较高；2. The solid phase carrier of the present invention uses hydrogel as a skeleton material, and functional groups are grafted on its surface, which can be used for enzymatic DNA synthesis, effectively reducing the steric hindrance of the enzymatic reaction process, and the synthesis efficiency is high;
3、本发明的制备固相载体的方法，操作简单，节能环保，所得固相载体舒展能力强、空间位阻低；3. The method for preparing a solid phase carrier of the present invention is simple to operate, energy-saving and environmentally friendly, and the obtained solid phase carrier has strong stretching ability and low steric hindrance;
4、本发明的固相载体用于酶促DNA合成可以降低酶促反应过程的空间位阻，极大程度地提高合成效率。4. The solid phase carrier of the present invention is used for enzymatic DNA synthesis to reduce the steric hindrance of the enzymatic reaction process and greatly improve the synthesis efficiency.
上述概述仅仅是为了说明书的目的，并不意图以任何方式进行限制。除上述描述的示意性的方面、实施方式和特征之外，通过参考附图和以下的详细描述，本申请进一步的方面、实施方式和特征将会是容易明白的。The above summary is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features of the present application will be readily apparent by reference to the accompanying drawings and the following detailed description.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
在附图中，除非另外规定，否则贯穿多个附图相同的附图标记表示相同或相似的部件或元素。这些附图不一定是按照比例绘制的。应该理解，这些附图仅描绘了根据本申请公开的一些实施方式，而不应将其视为是对本申请范围的限制。In the accompanying drawings, unless otherwise specified, the same reference numerals throughout the multiple drawings represent the same or similar parts or elements. These drawings are not necessarily drawn to scale. It should be understood that these drawings only depict some embodiments disclosed in the present application and should not be regarded as limiting the scope of the present application.
图1为本发明一实施例的制备固相载体的方法的流程图；FIG1 is a flow chart of a method for preparing a solid phase carrier according to an embodiment of the present invention;
图2为本发明一实施例的利用固相载体合成DNA的方法的流程图；FIG2 is a flow chart of a method for synthesizing DNA using a solid phase carrier according to an embodiment of the present invention;
图3为本发明实施例1中最终所得目标序列的测序结果；FIG3 is the sequencing result of the target sequence finally obtained in Example 1 of the present invention;
图4为本发明实施例2中最终所得目标序列的测序结果。FIG. 4 is the sequencing result of the target sequence finally obtained in Example 2 of the present invention.
具体实施方式DETAILED DESCRIPTION
为了更加清楚地理解本发明的技术特征、目的和有益效果，现对本发明的技术方案进行进一步的详细说明。在下文中，仅简单地描述了某些示例性实施例。正如本领域技术人员可认识到的那样，在不脱离本申请的精神或范围的情况下，可通过各种不同方式修改所描述的实施例。因此，附图和描述被认为本质上是示例性的而非限制性的。In order to more clearly understand the technical features, purposes and beneficial effects of the present invention, the technical scheme of the present invention is now further described in detail. Hereinafter, only some exemplary embodiments are simply described. As those skilled in the art will appreciate, the described embodiments may be modified in various ways without departing from the spirit or scope of the present application. Therefore, the drawings and descriptions are considered to be exemplary and non-restrictive in nature.
在本发明的一个方面，本发明提供了一种固相载体。该固相载体包括水凝胶骨架材料、功能性材料、交联剂以及添加剂；并且水凝胶骨架材料包括丙烯酸酯类高分子材料；添加剂包括增强剂、增塑剂以及光引发剂；在该固相载体的组分中，水凝胶骨架材料占各组分总质量的10%-35%。该固相载体至少具有以下有益效果的至少之一：舒展能力强、空间位阻低、合成效率较高。In one aspect of the present invention, the present invention provides a solid phase carrier. The solid phase carrier comprises a hydrogel skeleton material, a functional material, a crosslinking agent and an additive; and the hydrogel skeleton material comprises an acrylic polymer material; the additive comprises a reinforcing agent, a plasticizer and a photoinitiator; among the components of the solid phase carrier, the hydrogel skeleton material accounts for 10%-35% of the total mass of each component. The solid phase carrier has at least one of the following beneficial effects: strong stretching ability, low steric hindrance, and high synthesis efficiency.
为了方便理解，下面首先对该固相载体能够实现上述有益效果的原理进行简单说明：For ease of understanding, the following first briefly describes the principle by which the solid phase carrier can achieve the above beneficial effects:
水凝胶是一种具备特殊物理化学性质的聚合物材料，本发明的固相载体以水凝胶作为骨架材料，具备水凝胶的优秀物理化学性质，相对于传统的固相载体，具备溶胀而不溶解的特性。本发明通过在水凝胶表面接枝功能基团并改性，得到功能化水凝胶，得到空间位阻低、舒展能力强且适用范围广的固相载体。Hydrogel is a polymer material with special physical and chemical properties. The solid phase carrier of the present invention uses hydrogel as a skeleton material and has the excellent physical and chemical properties of hydrogel. Compared with traditional solid phase carriers, it has the characteristic of swelling but not dissolving. The present invention obtains functionalized hydrogel by grafting functional groups on the surface of hydrogel and modifying it, thereby obtaining a solid phase carrier with low steric hindrance, strong stretching ability and wide application range.
根据本发明的实施例，该固相载体包括水凝胶骨架材料、功能性材料、交联剂以及添加剂，并且进一步包括溶剂。其中，溶剂的种类不受特别的限制，可以为水和无机盐溶液，在具体实施方式中，优选地为水。此外，固相载体中水凝胶骨架材料、功能性材料、交联剂、添加剂以及溶剂的比例不受特别的限制，只要令水凝胶骨架材料占各组分总体积的10%-35%即可，例如在具体实施方式中，水凝胶骨架材料、功能性材料、交联剂、添加剂与溶剂的体积比可以为（3-10）：（2-5）：（0.1-1）：（5-10）：（4-8），优选地为（4-8）：（1-4）：（0.1-1）：（7-10）：（4-7）。According to an embodiment of the present invention, the solid phase carrier includes a hydrogel skeleton material, a functional material, a cross-linking agent and an additive, and further includes a solvent. The type of solvent is not particularly limited, and can be water and an inorganic salt solution. In a specific embodiment, it is preferably water. In addition, the proportion of the hydrogel skeleton material, the functional material, the cross-linking agent, the additive and the solvent in the solid phase carrier is not particularly limited, as long as the hydrogel skeleton material accounts for 10%-35% of the total volume of each component. For example, in a specific embodiment, the volume ratio of the hydrogel skeleton material, the functional material, the cross-linking agent, the additive and the solvent can be (3-10): (2-5): (0.1-1): (5-10): (4-8), preferably (4-8): (1-4): (0.1-1): (7-10): (4-7).
根据本发明的实施例，在形成固相载体的原料中，水凝胶骨架材料的种类不受特别的限制，例如，可以包括丙烯酸甲酯、丙烯酸乙酯、丙烯酸正丙酯、丙烯酸正丁酯、甲基丙烯酸羟乙酯、聚乙二醇二丙烯酸酯、甲基丙烯酸羟丙酯、甲基丙烯酸羟丁酯或其组合，优选地包括甲基丙烯酸羟乙酯、聚乙二醇二丙烯酸酯、甲基丙烯酸羟丙酯或其组合。功能性材料的种类也不受特别的限制，例如可以为碳水化合物，只要其上含有功能性基团即可，例如包括环氧基、伯胺、巯基、羰基、羟基、羧基或其组合的化合物，具体地，功能性材料包括甲基丙烯酸缩水甘油酯。交联剂包括乙二醇二丙烯酸酯。此外，添加剂是由增强剂、增塑剂与光引发剂构成的，并且在具体实施方式中，添加剂中增强剂、增塑剂与光引发剂的体积比为（1-5）：（3-6）：（0.1-1），优选地为（2-4）：（4-6）：（0.2-0.7）；其中，增强剂包括但不限于N-乙烯基吡咯烷酮，增塑剂包括但不限于乙二醇，光引发剂包括但不限于2-羟基-4’-(2-羟乙氧基)-2-甲基苯丙酮、2-羟基-2-甲基苯丙酮或其组合。According to an embodiment of the present invention, in the raw materials for forming the solid phase carrier, the type of hydrogel skeleton material is not particularly limited, for example, it can include methyl acrylate, ethyl acrylate, n-propyl acrylate, n-butyl acrylate, hydroxyethyl methacrylate, polyethylene glycol diacrylate, hydroxypropyl methacrylate, hydroxybutyl methacrylate or a combination thereof, preferably including hydroxyethyl methacrylate, polyethylene glycol diacrylate, hydroxypropyl methacrylate or a combination thereof. The type of functional material is also not particularly limited, for example, it can be a carbohydrate, as long as it contains a functional group, such as a compound including an epoxy group, a primary amine, a thiol group, a carbonyl group, a hydroxyl group, a carboxyl group or a combination thereof, specifically, the functional material includes glycidyl methacrylate. The crosslinking agent includes ethylene glycol diacrylate. In addition, the additive is composed of a reinforcing agent, a plasticizer and a photoinitiator, and in a specific embodiment, the volume ratio of the reinforcing agent, the plasticizer and the photoinitiator in the additive is (1-5): (3-6): (0.1-1), preferably (2-4): (4-6): (0.2-0.7); wherein the reinforcing agent includes but is not limited to N-vinyl pyrrolidone, the plasticizer includes but is not limited to ethylene glycol, and the photoinitiator includes but is not limited to 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, 2-hydroxy-2-methylpropiophenone or a combination thereof.
根据本发明的实施例，本发明的固相载体表面偶联有引发链，具体地，本发明的水凝胶骨架材料经过活化处理并在其表面偶联引发链。其中，水凝胶骨架材料是经过表面改性后进行活化处理的，表面改性包括在所述水凝胶骨架材料中引入氨基、羧基、环氧基、巯基以及乙烯基基团中的至少之一。经过表面改性以及活化处理的水凝胶骨架材料可以更好地偶联引发链，以实现酶促合成DNA的功能。According to an embodiment of the present invention, the surface of the solid phase carrier of the present invention is coupled with an initiator chain. Specifically, the hydrogel skeleton material of the present invention is activated and coupled with an initiator chain on its surface. The hydrogel skeleton material is activated after surface modification, and the surface modification includes introducing at least one of an amino group, a carboxyl group, an epoxy group, a thiol group and a vinyl group into the hydrogel skeleton material. The hydrogel skeleton material that has been surface modified and activated can better couple the initiator chain to achieve the function of enzymatic synthesis of DNA.
在本发明的另一方面，本发明提供了一种制备固相载体的方法。参考图1，该方法包括：将水凝胶骨架材料、功能性材料、交联剂、添加剂与溶剂混合后倒入模具中，在光照条件下照射制得水凝胶骨架；对水凝胶骨架表面进行改性处理得到改性水凝胶；对改性水凝胶进行活化处理后进行引发链偶联得到固相载体。其中，光照条件包括UV照射，光照时间为8-12分钟。该方法至少具有以下有益效果的至少之一：操作简单，节能环保，所得固相载体舒展能力强、空间位阻低。In another aspect of the present invention, the present invention provides a method for preparing a solid phase carrier. Referring to Figure 1, the method comprises: mixing a hydrogel skeleton material, a functional material, a cross-linking agent, an additive and a solvent and pouring the mixture into a mold, irradiating the mixture under light conditions to obtain a hydrogel skeleton; modifying the surface of the hydrogel skeleton to obtain a modified hydrogel; activating the modified hydrogel and then initiating chain coupling to obtain a solid phase carrier. The light conditions include UV irradiation, and the light exposure time is 8-12 minutes. The method has at least one of the following beneficial effects: simple operation, energy saving and environmental protection, and the obtained solid phase carrier has strong stretching ability and low steric hindrance.
关于水凝胶骨架材料、功能性材料、交联剂、添加剂与溶剂的种类，前面已经进行了详细的描述，在此不再赘述。The types of hydrogel skeleton materials, functional materials, cross-linking agents, additives and solvents have been described in detail above and will not be repeated here.
根据本发明的实施例，在将水凝胶骨架材料、功能性材料、交联剂、添加剂与溶剂混合的过程中，水凝胶骨架材料、功能性材料、交联剂、添加剂与溶剂的加入顺序不受特别的限制，只要最终形成均匀的预聚物即可。例如在具体实施方式中可以将预定比例的上述原料放入混合容器中，混匀，得到均聚物溶液；其中，混合容器的尺寸和材质均不受特别的限制，优选地为管，混合的方式也不受特别的限制，包括但不限于震荡、涡旋、翻转以及其他方式。而后，将所得预聚物溶液注入模具中，由于溶液内含有光引发剂，所以在光照的条件可以引发原料反应物之间的自由基发生聚合，得到成型的水凝胶骨架。其中，所用模具的材质、形状、尺寸均不受特别的限制，只要模具表面光滑平整即可。例如，在具体实施方式中，所用模具的材质包括但不限于塑料、玻璃、橡胶以及金属，并且优选地为塑料，例如热塑性塑料（包括PET材料等）；所用模具的形状只要便于将水凝胶骨架取出并具有一定高度即可，例如，可以为圆孔状或长方体状，并且优选地为圆孔状；所用模具的尺寸具体地可以根据所得预聚物溶液的体积决定，但是为节约制备原料，在具体实施方式中，所用模具的体积通常可以小于1cm3。此外，为避免制得的水凝胶载体过厚，使得后续还需进行切割处理，所以在具体实施方式中，模具的高度可以为，优选地为。综上所述，在具体实施方式中，本发明所用模具优选地为PET材料制得的、高度为的圆孔状模具。According to an embodiment of the present invention, in the process of mixing the hydrogel skeleton material, the functional material, the crosslinking agent, the additive and the solvent, the order of adding the hydrogel skeleton material, the functional material, the crosslinking agent, the additive and the solvent is not particularly limited, as long as a uniform prepolymer is finally formed. For example, in a specific embodiment, the above raw materials in a predetermined proportion can be put into a mixing container and mixed to obtain a homopolymer solution; wherein the size and material of the mixing container are not particularly limited, preferably tube, and the mixing method is not particularly limited, including but not limited to oscillation, vortex, flipping and other methods. Then, the obtained prepolymer solution is injected into the mold. Since the solution contains a photoinitiator, the free radicals between the raw material reactants can be induced to polymerize under the condition of light to obtain a shaped hydrogel skeleton. Among them, the material, shape and size of the mold used are not particularly limited, as long as the surface of the mold is smooth and flat. For example, in a specific embodiment, the material of the mold used includes but is not limited to plastic, glass, rubber and metal, and is preferably plastic, such as thermoplastic plastic (including PET material, etc.); the shape of the mold used is convenient for taking out the hydrogel skeleton and has a certain height, for example, it can be a circular hole shape or a rectangular parallelepiped shape, and is preferably a circular hole shape; the size of the mold used can be specifically determined according to the volume of the obtained prepolymer solution, but in order to save the preparation of raw materials, in a specific embodiment, the volume of the mold used can usually be less than 1 cm3. In addition, in order to avoid the obtained hydrogel carrier being too thick, so that subsequent cutting processing is required, the height of the mold can be in a specific embodiment. , preferably In summary, in a specific embodiment, the mold used in the present invention is preferably made of PET material with a height of A round hole shaped mold.
根据本发明的实施例，将上述预聚物溶液注入模具中后，可进一步用玻璃片压合，将气泡排出，而后使用UV进行照射以引发自由基聚合，形成稳定的水凝胶骨架。具体地，可以使用的UV灯进行照射，照射时间可以为，优选地为，其中，UV照射的方向不受特别的限制，但为避免局部光线过强，可以令UV光线与预聚物表面的角度为60-90°，并且优选地为90°垂直照射。而后将玻璃片取下，将水凝胶骨架从模具中取出并用纯水进行清洗，在纯水中保存以令其表面保持湿润，其中，玻璃片包括但不限于盖玻片。According to an embodiment of the present invention, after the prepolymer solution is injected into the mold, it can be further pressed with a glass sheet to expel the bubbles, and then irradiated with UV to induce free radical polymerization to form a stable hydrogel skeleton. The UV light can be used for irradiation. , preferably , wherein the direction of UV irradiation is not particularly limited, but to avoid excessive local light intensity, the angle between the UV light and the prepolymer surface can be 60-90°, and preferably 90° vertical irradiation. Then the glass sheet is removed, the hydrogel skeleton is taken out of the mold and washed with pure water, and stored in pure water to keep its surface moist, wherein the glass sheet includes but is not limited to a cover glass.
根据本发明的实施例，本发明进一步包括对所得水凝胶骨架表面进行改性处理，具体地，包括将上述水凝胶骨架从纯水中取出，而后用亲水改性化合物对其表面进行处理，以得到改性水凝胶。其中，亲水改性化合物的种类不受特别的限制，具体地，可以根据选用的上述功能性材料的种类决定，例如，在具体实施方式中，亲水改性化合物可以包括富含氨基的表面改性材料、富含羧基的功能材料以及富含环氧基的功能材料中的至少之一。其中，富含氨基的表面改性材料、富含羧基的功能材料以及富含环氧基的功能材料的种类均不受特别的限制，只要具有改性作用即可。例如，在具体实施方式中，富含氨基的表面改性材料包括但不限于支化聚乙烯亚胺。并且，由于聚谷氨酸的分子链上有大量的游离羧基，作为富含羧基的功能材料可以为水凝胶表面提供大量羧基，可以实现对生物大分子的高效偶联；以及聚乙二醇是具有优良水溶性的线性分子，采用聚乙二醇的衍生物作为富含羧基的功能材料可以有效降低表面的偶联密度，降低反应位阻；所以富含羧基的功能材料优选地包括聚谷氨酸、聚乙二醇衍生物或其组合，其中，聚乙二醇衍生物优选地包括氨基聚乙二醇羧基。此外，富含环氧基的功能材料包括但不限于聚乙二醇二缩水甘油醚。According to an embodiment of the present invention, the present invention further includes modifying the surface of the obtained hydrogel skeleton, specifically, taking the above hydrogel skeleton out of pure water, and then treating its surface with a hydrophilic modification compound to obtain a modified hydrogel. Wherein, the type of hydrophilic modification compound is not particularly limited, and specifically, it can be determined according to the type of the above functional material selected. For example, in a specific embodiment, the hydrophilic modification compound can include at least one of an amino-rich surface modification material, a carboxyl-rich functional material, and an epoxy-rich functional material. Wherein, the types of amino-rich surface modification materials, carboxyl-rich functional materials, and epoxy-rich functional materials are not particularly limited, as long as they have a modification effect. For example, in a specific embodiment, the amino-rich surface modification material includes but is not limited to branched polyethyleneimine. In addition, since there are a large number of free carboxyl groups on the molecular chain of polyglutamic acid, as a carboxyl-rich functional material, a large number of carboxyl groups can be provided to the hydrogel surface, and efficient coupling of biomacromolecules can be achieved; and polyethylene glycol is a linear molecule with excellent water solubility, and the use of polyethylene glycol derivatives as carboxyl-rich functional materials can effectively reduce the surface coupling density and reduce the reaction steric hindrance; therefore, the carboxyl-rich functional material preferably includes polyglutamic acid, polyethylene glycol derivatives or a combination thereof, wherein the polyethylene glycol derivative preferably includes amino polyethylene glycol carboxyl. In addition, epoxy-rich functional materials include but are not limited to polyethylene glycol diglycidyl ether.
根据本发明的实施例，在进行改性处理的过程中，处理次数不受特别的限制，具体地，可以进行一次或多次处理，并且每次处理所用的亲水改性化合物的种类可以相同或不相同；处理的时间也不受特别的限制，优选地处理时间大于；此外，所用亲水改性化合物的浓度也不受特别的限制，但为了避免浓度过高造成浪费，或者浓度过低使得反应缓慢，所以在具体实施方式中，亲水改性化合物的浓度优选地为。此外，本发明可进一步包括在每次改性处理之后将水凝胶取出并对其进行清洗或在PBS缓冲液中储存，而后再进行下一次处理，并且将所得改性水凝胶在纯水中保存以令其表面保持湿润。According to an embodiment of the present invention, during the modification process, the number of treatments is not particularly limited. Specifically, one or more treatments may be performed, and the types of hydrophilic modification compounds used in each treatment may be the same or different. The treatment time is also not particularly limited. Preferably, the treatment time is greater than In addition, the concentration of the hydrophilic modification compound is not particularly limited, but in order to avoid waste due to too high a concentration or slow reaction due to too low a concentration, in a specific embodiment, the concentration of the hydrophilic modification compound is preferably In addition, the present invention may further include taking out the hydrogel after each modification treatment and washing it or storing it in a PBS buffer before performing the next treatment, and storing the obtained modified hydrogel in pure water to keep its surface moist.
根据本发明的实施例，本发明进一步包括对上述改性水凝胶表面进行活化处理，具体地，包括将上述改性水凝胶从纯水中取出，用活化化合物对其进行处理。其中，所用的活化化合物种类不受特别的限制，例如可以包括碳二亚胺类羧基活化剂、NHS活化脂中间物或其组合，优选地包括碳二亚胺类羧基活化剂和NHS活化脂中间物，其中，碳二亚胺类羧基活化剂包括但不限于1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐和二环己基碳二亚胺中的至少之一；NHS活化脂中间物可包括N-羟基硫代琥珀酰亚胺钠盐、N-羟基琥珀酰亚胺或其组合。处理的时间也不受特别的限制，优选地为，更优选地为。此外，所用活化化合物的浓度也不受特别的限制，但为了避免浓度过高造成浪费，或者浓度过低使得反应缓慢，所以在具体实施方式中，活化化合物的浓度优选地为。此外，本发明可进一步包括在完成活化处理后将所得活化的改性水凝胶置于MES缓冲液中储存，而后再进行后续处理。According to an embodiment of the present invention, the present invention further includes activating the surface of the modified hydrogel, specifically, taking the modified hydrogel out of pure water and treating it with an activating compound. The type of activating compound used is not particularly limited, and may include, for example, a carbodiimide carboxyl activator, an NHS-activated lipid intermediate, or a combination thereof, preferably a carbodiimide carboxyl activator and an NHS-activated lipid intermediate, wherein the carbodiimide carboxyl activator includes, but is not limited to, at least one of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and dicyclohexylcarbodiimide; the NHS-activated lipid intermediate may include N-hydroxysulfosuccinimide sodium salt, N-hydroxysuccinimide, or a combination thereof. The treatment time is also not particularly limited, and is preferably , more preferably In addition, the concentration of the activated compound used is not particularly limited, but in order to avoid waste caused by too high a concentration or slow reaction caused by too low a concentration, in a specific embodiment, the concentration of the activated compound is preferably In addition, the present invention may further include storing the activated modified hydrogel obtained after the activation treatment is completed in MES buffer, and then performing subsequent treatment.
根据本发明的实施例，本发明进一步包括在所得活化的改性水凝胶表面偶联引发链，具体地，包括将经过活化处理的改性水凝胶浸入链霉亲和素偶联溶液中，在常温条件下震荡反应过夜，令链霉亲和素在改性水凝胶表面通过酰胺化反应偶联在改性水凝胶表面，将其置于PBS缓冲液中储存，将其取出后再浸入引发链偶联溶液中，得到固相载体。其中，链霉亲和素偶联溶液中包括链霉亲和素。引发链偶联溶液包括引发链。此外，在具体实施方式中，引发链包括5’端生物素标记的DNA序列，以利用链霉亲和素-生物素的亲和反应偶联引发链，得到偶联有引发链的固相载体，并将其置于Tris-HCl缓冲液中储存，用于后续使用。According to an embodiment of the present invention, the present invention further includes coupling the initiator chain on the surface of the activated modified hydrogel obtained, specifically, immersing the modified hydrogel after the activation treatment in a streptavidin coupling solution, shaking the reaction overnight at room temperature, allowing the streptavidin to couple to the surface of the modified hydrogel through an amidation reaction, storing it in a PBS buffer, taking it out and then immersing it in the initiator chain coupling solution , to obtain a solid phase carrier. The streptavidin coupling solution includes Streptavidin. Primer Chain Coupling Solution includes In a specific embodiment, the initiator chain includes a DNA sequence labeled with biotin at the 5' end, and the initiator chain is coupled by a streptavidin-biotin affinity reaction to obtain a solid phase carrier coupled with the initiator chain, which is then stored in a Tris-HCl buffer for subsequent use.
在本发明的又一方面，本发明提供了一种利用上述固相载体合成DNA的方法，参考图2，该方法包括以下步骤：裁剪固相载体并利用其进行DNA合成反应和脱保护反应，不断重复进行合成反应与脱保护反应循环，以合成目标序列；对经合成反应与脱保护反应循环后的固相载体进行解离反应；以解离反应得到的解离产物为模板进行PCR扩增；对PCR扩增得到的产物进行琼脂糖凝胶电泳，回收目标电泳条带并对其进行测序分析。该方法至少具有以下有益效果的至少之一：降低酶促反应过程的空间位阻，极大程度地提高合成效率。In another aspect of the present invention, the present invention provides a method for synthesizing DNA using the above-mentioned solid phase carrier. Referring to FIG2 , the method comprises the following steps: cutting the solid phase carrier and using it to perform DNA synthesis reaction and deprotection reaction, and continuously repeating the synthesis reaction and deprotection reaction cycle to synthesize the target sequence; performing a dissociation reaction on the solid phase carrier after the synthesis reaction and deprotection reaction cycle; performing PCR amplification using the dissociation product obtained by the dissociation reaction as a template; performing agarose gel electrophoresis on the product obtained by PCR amplification, recovering the target electrophoresis band and performing sequencing analysis on it. The method has at least one of the following beneficial effects: reducing the steric hindrance of the enzymatic reaction process and greatly improving the synthesis efficiency.
根据本发明的实施例，本发明进一步包括对固相载体进行裁剪，裁剪后固相载体的形状、尺寸不受特别的限制，只要可以放入PCR管中进行合成反应即可。具体地，可以将固相载体裁剪成宽（1.2-2.5）mm，长（2.5-3.5）mm的长方形，并且将该固相载体浸入到DNA酶促合成反应液中进行DNA合成反应，其中，DNA合成反应是在30℃-45℃的温度条件下进行的，反应时间可以为，优选地为，反应结束后在4℃下保存。其中，DNA酶促合成反应液包括末端脱氧核苷酸转移酶、末端保护碱基单体、氯化钴、PBS和水，各组分具体含量如下表1所示，其中，末端保护碱基单体包括氨氧基保护的三磷酸脱氧鸟苷、氨氧基保护的三磷酸脱氧腺苷、氨氧基保护的三磷酸脱氧胞苷、氨氧基保护的三磷酸脱氧胞苷，并且每次合成反应中反应液体系仅包括一种末端保护碱基单体。此外，DNA合成反应可以在金属浴、水浴或者PCR仪中进行，优选地在PCR仪中进行。According to an embodiment of the present invention, the present invention further includes cutting the solid phase carrier. The shape and size of the solid phase carrier after cutting are not particularly limited, as long as it can be placed in a PCR tube for synthesis reaction. Specifically, the solid phase carrier can be cut into a rectangle with a width of (1.2-2.5) mm and a length of (2.5-3.5) mm, and the solid phase carrier can be immersed in a DNA enzyme synthesis reaction solution for DNA synthesis reaction, wherein the DNA synthesis reaction is carried out at a temperature of 30°C-45°C, and the reaction time can be , preferably , stored at 4°C after the reaction. The DNA enzymatic synthesis reaction solution includes terminal deoxynucleotidyl transferase, terminal protection base monomer, cobalt chloride, PBS and water, and the specific content of each component is shown in Table 1 below, wherein the terminal protection base monomer includes aminooxy-protected deoxyguanosine triphosphate, aminooxy-protected deoxyadenosine triphosphate, aminooxy-protected deoxycytidine triphosphate, and aminooxy-protected deoxycytidine triphosphate, and the reaction solution system includes only one terminal protection base monomer in each synthesis reaction. In addition, the DNA synthesis reaction can be carried out in a metal bath, a water bath or a PCR instrument, preferably in a PCR instrument.
根据本发明的实施例，本发明进一步包括对上述DNA合成反应后的固相载体进行脱保护反应，具体地，包括用镊子将上述DNA合成反应完成后的固相载体取出，用纯水冲洗后浸入到脱保护反应液中，浸没后取出固相载体并用纯水进行冲洗。其中，固相载体与脱保护反应液加入到PCR管的顺序不受特别的限制，可以先将固相载体放于PCR管中，也可先将脱保护反应液加入到PCR管中，在具体实施方式中，优选地先将固相载体放于PCR管中，而后加入脱保护反应液。此外，脱保护反应液包括亚硝酸钠和冰乙酸，具体地，脱保护反应液是通过将45-50mg亚硝酸钠溶于纯水中，而后加入1%冰乙酸溶液制得的。According to an embodiment of the present invention, the present invention further comprises performing a deprotection reaction on the solid phase carrier after the DNA synthesis reaction, specifically, taking out the solid phase carrier after the DNA synthesis reaction with tweezers, rinsing it with pure water and immersing it in a deprotection reaction solution, and immersing it in The solid phase carrier is then taken out and rinsed with pure water. The order in which the solid phase carrier and the deprotection reaction solution are added to the PCR tube is not particularly limited. The solid phase carrier can be placed in the PCR tube first, or the deprotection reaction solution can be added to the PCR tube first. In a specific embodiment, the solid phase carrier is preferably placed in the PCR tube first, and then the deprotection reaction solution is added. In addition, the deprotection reaction solution includes sodium nitrite and glacial acetic acid. Specifically, the deprotection reaction solution is prepared by dissolving 45-50 mg of sodium nitrite in In pure water, then add Prepared from 1% glacial acetic acid solution.
根据本发明的实施例，本发明进一步包括不断重复上述合成反应与脱保护反应。具体地，可以按照目标序列的碱基序列顺序加入末端保护碱基单体进行酶促DNA合成反应，重复DNA合成反应-脱保护反应循环，直至合成目标序列。According to an embodiment of the present invention, the present invention further includes continuously repeating the above synthesis reaction and deprotection reaction. Specifically, terminal protection base monomers can be added in the order of the base sequence of the target sequence to carry out enzymatic DNA synthesis reaction, and the DNA synthesis reaction-deprotection reaction cycle is repeated until the target sequence is synthesized.
根据本发明的实施例，本发明进一步包括对上述进行DNA合成反应-脱保护反应循环后的固相载体进行解离反应，以将合成的目标序列解离下来。具体地，可以将合成反应与脱保护反应循环后的固相载体浸入到纯水中，在80℃-100℃的温度条件下反应得到解离产物，反应结束后在4℃下保存。According to an embodiment of the present invention, the present invention further comprises performing a dissociation reaction on the solid phase carrier after the DNA synthesis reaction-deprotection reaction cycle to dissociate the synthesized target sequence. Specifically, the solid phase carrier after the synthesis reaction and deprotection reaction cycle can be immersed in pure water and reacted at a temperature of 80°C-100°C. The dissociation product was obtained and stored at 4°C after the reaction.
根据本发明的实施例，在进行解离反应之前，本发明可进一步包括在合成的目标序列的3’末端继续合成末端无保护的碱基单体，以便于合成引物序列进行后续测序反应，其中末端无保护的碱基单体包括但不限于dATP单体。例如，在具体实施方式中，可以对经合成反应与脱保护反应循环后的固相载体进行PolyA合成反应，将该固相载体浸入到PolyA合成反应液中，在30℃-45℃的温度条件下反应令目标序列的3’端合成PolyA片段，反应结束后在4℃下保存，而后用镊子将完成PolyA合成反应的水凝胶取出，经纯水冲洗后放入新的PCR管中，用于后续解离反应。其中，PolyA合成反应液包括末端脱氧核苷酸转移酶、无保护的dATP单体、缓冲液和水，具体地合成体系如下表2所示。此外，PolyA合成反应可以在金属浴、水浴或者PCR仪中进行，优选地在PCR仪中进行。According to an embodiment of the present invention, before the dissociation reaction is performed, the present invention may further include continuing to synthesize a terminal unprotected base monomer at the 3' end of the synthesized target sequence to facilitate the synthesis of the primer sequence for subsequent sequencing reaction, wherein the terminal unprotected base monomer includes but is not limited to a dATP monomer. For example, in a specific embodiment, a PolyA synthesis reaction may be performed on a solid phase support after a synthesis reaction and a deprotection reaction cycle, and the solid phase support may be immersed in a PolyA synthesis reaction solution and reacted at a temperature of 30°C-45°C. The 3' end of the target sequence is synthesized into a PolyA fragment, which is stored at 4°C after the reaction is completed, and then the hydrogel that has completed the PolyA synthesis reaction is taken out with tweezers, rinsed with pure water, and placed in a new PCR tube for subsequent dissociation reaction. The PolyA synthesis reaction solution includes terminal deoxynucleotidyl transferase, unprotected dATP monomer, buffer and water, and the specific synthesis system is shown in Table 2 below. In addition, the PolyA synthesis reaction can be carried out in a metal bath, a water bath or a PCR instrument, preferably in a PCR instrument.
根据本发明的实施例，本发明可进一步包括对解离反应产生的解离产物进行检测，包括对解离产物进行PCR扩增，并对扩增得到的PCR产物进行琼脂糖凝胶电泳检测，对跑胶条带进行试剂盒回收，对回收产物进行测序分析。其中，PCR扩增的反应体系和反应条件不受特别的限制，具体地，反应体系可根据所选用的DNA聚合酶的种类决定，而反应条件可根据目标序列的长度、引物序列的解链温度以及DNA聚合酶的延伸速度等来调整。在具体实施方式中，所选用的PCR扩增的反应体系如下表3所示。According to an embodiment of the present invention, the present invention may further include detecting the dissociation products generated by the dissociation reaction, including PCR amplification of the dissociation products, and agarose gel electrophoresis detection of the amplified PCR products, kit recovery of the gel strips, and sequencing analysis of the recovered products. Among them, the reaction system and reaction conditions of PCR amplification are not particularly limited. Specifically, the reaction system can be determined according to the type of DNA polymerase selected, and the reaction conditions can be adjusted according to the length of the target sequence, the melting temperature of the primer sequence, and the extension speed of the DNA polymerase. In a specific embodiment, the selected PCR amplification reaction system is shown in Table 3 below.
该固相载体可用于包括但不限于DNA酶促合成，可以在固相载体表面表面接枝不同的功能性基团，制备出适用于各项工作的功能化固相载体。The solid phase carrier can be used for, including but not limited to, DNA enzymatic synthesis, and different functional groups can be grafted onto the surface of the solid phase carrier to prepare a functionalized solid phase carrier suitable for various tasks.
实施例Example
下面通过具体实施例对本发明提出的方法进行详细说明。下述实施例中所用方法如无特别说明均为常规方法，所用的试剂如无特别说明均为可商购的试剂。The method proposed in the present invention is described in detail below by means of specific examples. The methods used in the following examples are conventional methods unless otherwise specified, and the reagents used are commercially available reagents unless otherwise specified.
实施例1Example 1
1. 制备水凝胶骨架1. Preparation of hydrogel skeleton
取下述形成固相载体的原料于管中，涡旋摇匀，制得预聚物溶液：Take the following raw materials to form a solid phase carrier Tube, vortex and shake to prepare prepolymer solution:
水凝胶骨架材料：甲基丙烯酸羟乙酯，；Hydrogel skeleton material: Hydroxyethyl methacrylate, ;
功能性材料：甲基丙烯酸缩水甘油酯，；Functional materials: Glycidyl methacrylate, ;
交联剂：乙二醇二丙烯酸酯；；Crosslinking agent: ethylene glycol diacrylate; ;
添加剂：增强剂——N-乙烯基吡咯烷酮，；Additives: Enhancer - N-vinyl pyrrolidone, ;
增塑剂——乙二醇，；Plasticizer - Ethylene glycol, ;
光引发剂——2-羟基-2-甲基苯丙酮，。Photoinitiator - 2-hydroxy-2-methylpropiophenone, .
将所得均匀的预聚物溶液倾倒在PET材质的圆孔模具中，使用盖玻片压合，将气泡排出，使用灯点光源90°垂直照射10min，之后将盖玻片揭开，将水凝胶骨架取出并用纯水清洗，放于纯水中储存备用。本实施例所用模具的直径为，厚度为。Pour the obtained uniform prepolymer solution into a round hole mold made of PET material, press it with a cover glass to remove the bubbles, and use The light source was irradiated vertically at 90° for 10 min, and then the cover glass was removed, the hydrogel skeleton was taken out and washed with pure water, and then stored in pure water for later use. The diameter of the mold used in this embodiment is , thickness is .
2. 制备改性水凝胶2. Preparation of modified hydrogels
氨基功能化：称取分子量支化聚乙烯亚胺溶解于溶液中，将水凝胶骨架浸入到该溶液中，常温下震荡反应2小时后取出，清水冲洗后，置于缓冲液中暂存。Amino functionalization: weigh Molecular weight branched polyethyleneimine dissolved in The hydrogel skeleton was immersed in the solution, shaken and reacted at room temperature for 2 hours, then taken out, rinsed with water, and placed Store in buffer.
羧基功能化：称取分子量聚谷氨酸溶解于缓冲液中，常温震荡溶解，待溶液澄清后，向溶液中加入碳二亚胺盐酸盐、，继续溶解后得到反应液，将水凝胶骨架从缓冲液中取出，放入上述反应液中反应3小时后取出，制得改性水凝胶，将其置于纯水中备用。Carboxyl functionalization: weigh Molecular weight polyglutamic acid dissolved in Buffer solution, shake at room temperature to dissolve, wait for the solution to become clear, add Carbodiimide hydrochloride, , and then continue to dissolve to obtain a reaction solution, and then separate the hydrogel skeleton from The modified hydrogel was taken out from the buffer solution, placed in the above reaction solution for reaction for 3 hours, and then taken out to obtain the modified hydrogel, which was placed in pure water for later use.
3. 制得固相载体3. Preparation of solid phase carrier
活化处理：称取碳二亚胺盐酸盐、溶解于缓冲液中，将所得改性水凝胶浸入到该溶液中反应后取出，此时改性水凝胶的表面活化完成，在缓冲液中暂存。Activation treatment: weigh Carbodiimide hydrochloride, Dissolved in The modified hydrogel was immersed in the solution to react After being taken out, the surface activation of the modified hydrogel is completed. Store in buffer.
偶联引发链：称取链霉亲和素溶解于缓冲液中，立即将表面活化完成的改性水凝胶浸入到该溶液中，常温震荡反应过夜后取出，通过酰胺化反应在水凝胶表面完成链霉亲和素偶联，将所得固相载体置于缓冲液中暂存。而后将CTTCAATCAGTCTAGCTCAATAT）溶解于缓冲液中，将完成偶联链霉亲和素的水凝胶浸入到该溶液中常温震荡反应后取出，通过链霉亲和素-生物素的亲和反应原理，在水凝胶表面完成引发链的偶联，制得固相载体，将所得固相载体放入缓冲液中备用。Coupling initiation chain: weigh Streptavidin dissolved in The modified hydrogel with surface activation was immediately immersed in the solution, shaken and reacted at room temperature overnight, and then taken out. Streptavidin was coupled on the surface of the hydrogel through amidation reaction, and the obtained solid phase carrier was placed on Store in buffer temporarily. CTTCAATCAGTCTAGCTCAATAT) dissolved in The streptavidin-coupled hydrogel is immersed in the buffer solution and shaken at room temperature for reaction. After that, the initiator chain is coupled on the hydrogel surface through the affinity reaction principle of streptavidin-biotin to obtain a solid phase carrier. Buffer for later use.
4. DNA合成反应4. DNA Synthesis Reaction
将所得固相载体从Tris-HCl缓冲液中取出，裁剪成宽，长的方形，浸入到装载如下试剂的反应体系的PCR管中，在PCR仪中进行酶促DNA合成，其中，酶促DNA合成反应体系和PCR仪工作条件分别如下表4和表5所示：The solid phase carrier was removed from the Tris-HCl buffer and cut into ,long The square is immersed in a PCR tube containing the following reagents in the reaction system, and enzymatic DNA synthesis is performed in a PCR instrument, wherein the enzymatic DNA synthesis reaction system and the PCR instrument working conditions are shown in Table 4 and Table 5 respectively:
酶促DNA合成完成后，用镊子夹出水凝胶，用纯水冲洗后将其放入新的PCR管中，用于后续脱保护反应。After the enzymatic DNA synthesis is completed, the hydrogel is taken out with tweezers, rinsed with pure water and placed in a new PCR tube for subsequent deprotection reaction.
5. 脱保护反应5. Deprotection reaction
称取47.2mg 亚硝酸钠溶于纯水，加入冰乙酸溶液，得到脱保护反应液，将该脱保护反应液加入到上述装有经过末端保护碱基合成的水凝胶的PCR管中，浸没反应后取出，用纯水冲洗后，重复步骤4和步骤5中DNA合成反应-脱保护反应循环，按照CGTCGTCGTC的顺序进行多碱基的合成。Weigh 47.2 mg of sodium nitrite and dissolve it in Pure water, add The deprotection reaction solution was added into the PCR tube containing the hydrogel synthesized with terminal protection bases, and the reaction mixture was immersed in the PCR tube. Then take it out, rinse it with pure water, and repeat the DNA synthesis reaction-deprotection reaction cycle in steps 4 and 5 to synthesize multiple bases in the order of CGTCGTCGTC.
6. PolyA合成6. PolyA Synthesis
上述DNA合成反应-脱保护反应循环完成后，进行PolyA合成，在目标序列的3’-末端加入PolyA片段进行PCR扩增，对合成结果进行表征。PolyA的合成体系如下表6所示，将DNA合成反应-脱保护反应循环完成的水凝胶浸入到该合成体系后，在PCR仪中进行PolyA合成，PCR仪工作条件如下表7所示。After the DNA synthesis reaction-deprotection reaction cycle is completed, PolyA synthesis is performed, and a PolyA fragment is added to the 3'-end of the target sequence for PCR amplification, and the synthesis result is characterized. The synthesis system of PolyA is shown in Table 6 below, and after the hydrogel that has completed the DNA synthesis reaction-deprotection reaction cycle is immersed in the synthesis system, PolyA synthesis is performed in a PCR instrument, and the working conditions of the PCR instrument are shown in Table 7 below.
PolyA合成合成完成后，用镊子夹出固相载体，用纯水冲洗后放入新的PCR管中，用于后续解离反应。After the PolyA synthesis is completed, the solid phase carrier is removed with tweezers, rinsed with pure water and placed in a new PCR tube for subsequent dissociation reaction.
7. 解离反应7. Dissociation reaction
向装有PolyA合成完成的固相载体的PCR管中加入纯水，在PCR仪上进行热解离反应，PCR仪工作条件如下表8所示：Add the following to the PCR tube containing the solid phase carrier after the synthesis of PolyA: Pure water, thermal dissociation reaction was performed on a PCR instrument. The working conditions of the PCR instrument are shown in Table 8 below:
8. 解离产物检测8. Detection of dissociation products
PCR扩增：对解离反应得到的解离产物进行PCR扩增操作，PCR的反应体系如下表9所示，其中，所用模板为解离产物，首引物序列为；尾引物序列为，并且PCR反应条件如下表10所示。PCR amplification: The dissociation product obtained by the dissociation reaction was subjected to PCR amplification. The PCR reaction system is shown in Table 9 below, wherein the template used is the dissociation product, and the first primer sequence is The tail primer sequence is , and the PCR reaction conditions are shown in Table 10 below.
对PCR扩增产物进行琼脂糖凝胶电泳分子，切割跑胶所得目的条带，使用胶回收试剂盒对其进行回收，将回收得到的DNA产物进行测序分析，对PolyA前的碱基数与初始引发链进行对比，观察目标序列的合成情况，计算最终合成效率。测序结果如图3所示，5条有效数据中具备合成能力的序列共3条，合成效率100%，全部成功。The PCR amplification product was subjected to agarose gel electrophoresis, the target band obtained by running the gel was cut, and it was recovered using a gel recovery kit. The recovered DNA product was sequenced and analyzed, the number of bases before PolyA was compared with the initial priming chain, the synthesis of the target sequence was observed, and the final synthesis efficiency was calculated. The sequencing results are shown in Figure 3. Among the 5 valid data, there are 3 sequences with synthesis ability, the synthesis efficiency is 100%, and all are successful.
实施例2Example 2
1. 制备水凝胶骨架1. Preparation of hydrogel skeleton
与实施例1相同。Same as Example 1.
2. 制备改性水凝胶2. Preparation of modified hydrogels
氨基功能化：称取分子量支化聚乙烯亚胺溶解于溶液中，将水凝胶骨架浸入到该溶液中，常温下震荡反应2小时后取出，清水冲洗后，置于缓冲液中暂存。Amino functionalization: weigh Molecular weight branched polyethyleneimine dissolved in The hydrogel skeleton was immersed in the solution, shaken and reacted at room temperature for 2 hours, then taken out, rinsed with water, and placed Store in buffer.
环氧基功能化：称取分子量聚乙二醇二缩水甘油醚溶解于缓冲液中，常温震荡溶解，待溶液澄清后，将水凝胶骨架从缓冲液中取出，浸入到该溶液中反应过夜后取出，置于缓冲液中暂存。Epoxy functionalization: weigh Molecular weight Polyethylene glycol diglycidyl ether is dissolved in Buffer solution, shake and dissolve at room temperature, wait for the solution to become clear, remove the hydrogel skeleton from Take it out from the buffer solution, immerse it in the solution to react overnight, then take it out and place it Store in buffer.
羧基功能化：称取分子量氨基聚乙二醇羧基溶解于溶液中，将水凝胶骨架从缓冲液中取出，浸入到该溶液中，反应过夜后取出，置于纯水中备用。Carboxyl functionalization: weigh Molecular weight amino polyethylene glycol carboxyl dissolved in In the solution, the hydrogel skeleton is Take it out from the buffer solution, immerse it in this solution, take it out after reacting overnight, and place it in pure water for later use.
3. 制得固相载体3. Preparation of solid phase carrier
与实施例1相同。Same as Example 1.
4. DNA合成反应4. DNA Synthesis Reaction
与实施例1相同。Same as Example 1.
5. 脱保护反应5. Deprotection reaction
与实施例1相同。Same as Example 1.
6. PolyA合成6. PolyA Synthesis
与实施例1相同。Same as Example 1.
7. 解离反应7. Dissociation reaction
与实施例1相同。Same as Example 1.
8. 解离产物检测8. Detection of dissociation products
检测方法与实施例1相同。测序结果如图4所示，6条有效数据中具备合成能力的序列共6条，4条存在1-2个碱基缺失，2条全部正确。The detection method is the same as that in Example 1. The sequencing results are shown in FIG4 . Among the 6 valid data, there are 6 sequences with synthesis ability, 4 of which have 1-2 base deletions, and 2 are all correct.
总的来说，本发明提出的固相载体，以水凝胶作为骨架材料，具备水凝胶的优秀物理化学性质，具备溶胀而不溶解的特性。本发明通过在水凝胶表面接枝功能基团并改性，得到功能化水凝胶，得到空间位阻低、舒展能力强且适用范围广的固相载体。此外，利用本发明的固相载体可进行酶促DNA合成反应，且合成的准确率较高，合成效率也较高。In general, the solid phase carrier proposed in the present invention uses hydrogel as a skeleton material, has excellent physical and chemical properties of hydrogel, and has the characteristics of swelling but not dissolving. The present invention obtains functionalized hydrogel by grafting functional groups on the surface of hydrogel and modifying it, thereby obtaining a solid phase carrier with low steric hindrance, strong stretching ability and wide application range. In addition, the solid phase carrier of the present invention can be used to carry out enzymatic DNA synthesis reaction, and the synthesis accuracy is high and the synthesis efficiency is also high.
以上显示和描述了本发明的基本原理和主要特征和本发明的优点，对于本领域技术人员而言，显然本发明不限于上述示范性实施例的细节，而且在不背离本发明的精神或基本特征的情况下，能够以其他的具体形式实现本发明。因此，无论从哪一点来看，均应将实施例看作是示范性的，而且是非限制性的。The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive.
此外，应当理解，虽然本说明书按照实施方式加以描述，但并非每个实施方式仅包含一个独立的技术方案，说明书的这种叙述方式仅仅是为清楚起见，本领域技术人员应当将说明书作为一个整体，各实施例中的技术方案也可以经适当组合，形成本领域技术人员可以理解的其他实施方式。In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311029717.4ACN117024778A (en) | 2023-08-16 | 2023-08-16 | Solid phase carrier for enzymatic DNA synthesis and preparation method and application thereof |
| Application Number | Priority Date | Filing Date | Title |
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| CN202311029717.4ACN117024778A (en) | 2023-08-16 | 2023-08-16 | Solid phase carrier for enzymatic DNA synthesis and preparation method and application thereof |
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| CN117024778Atrue CN117024778A (en) | 2023-11-10 |
| Application Number | Title | Priority Date | Filing Date |
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| CN202311029717.4APendingCN117024778A (en) | 2023-08-16 | 2023-08-16 | Solid phase carrier for enzymatic DNA synthesis and preparation method and application thereof |
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